Our objective is to elucidate the structure,function, and mechanism of the ubiquinone (Q)-mediated electron transfer complexes of the mitochondrial respiratory chain. During past support periods we have established protocols for site-directed mutagenesis coupled with in vivo and in vitro reconstitutionto study some of the structural and functional relationships in succinate-Q reductase (SQR) and ubiquinol-cytochromec reductase (QCR). We have crystallized QCR, solved its structure to 2.9 A resolution and indicated that QCR is a functional dimer and that the catalytic cycle of the complex involves movement of the head domain of iron-sulfur protein (ISP). Functional evidence for such movement was obtained from characterization of Rhodobacter sphaeroides bc\ complexes with altered ISP necks. In addition to electron and proton transfer, we have discovered and studied two more functions of QCR, superoxide radical generation and mitochondrial processing peptidase activity (MPP). In the next grant period we will focus on the elucidation of the mechanism of electron transfer and proton translocationin QCR and of electron transfer in SQR. The approaches to be used include kinetic rate determinations, various spectroscopic measurements, organic synthesis, resolution and reconstitution, molecular genetics, and protein crystallography. The specific aims are: (A) to obtain structuraldata for mechanistic studies, including the Q-binding at the Qo site pocket, the proton transfer path, structure water, hydrogen bonding, and the interaction domain between cytochromes c\ and c. This will be achieved by obtaining high resolution QCR structure and by X-ray analysis of QCR crystals in different redox states, in the presence of non-oxidizable Q derivatives, or with cytochrome c; (B) to elucidate the reaction mechanisms for electron and proton transfer in QCR. This will be achieved by investigating the movement of the head domain of ISP via functional analysis of mutantshaving an intersubunitcystine bridge, salt bridge, or hydrogen bond at the interface between cytochrome b and ISP, and via the conformational changes of cytochrome b or c1? at the interface with ISP, during the oxidation-reduction cycle or binding with different Qo site inhibitors; by determining the acid induced intra-molecular electron transfer rates between 2Fe2S and heme ct using a proton caged compound activated by pulse laser; and by probing the nature of the intra-molecularpath of QH2 in QCR by site directed mutagenesis; (C) to synthesize Q-derivatives and inhibitors for protein:Q interaction studies; (D) to elucidate electron transfer mechanisms in SQR by establishing the identity of QPs2; reconstitution of SQR from recombinantQPs subunits and isolated SDH; identification of SDH docking site(s) in QPs subunits;and crystallization and 3-D structural analysis of mitochondrial SQR. Successful elucidation of the reaction mechanismsfor electron and/or proton transfer of SQR and QCR will provide information crucial to understandingthe mitochondrialbioenergetic process and thus help in drug design for mitochondrial related diseases. Also, since the quinonereactive sites are responsible for superoxide generation and Q can scavenge singlet oxygen, detailed knowledge of the structure-function relationships and reaction mechanisms of Q- mediated electron transfer should provide valuable informationfor the study of oxidative stress. This information will be useful in pharmaceutical investigations of cytotoxicityand the aging process. PERFORMANCE S1TE(S) (organization, city, state) Department of Biochemistry & Molecular Biology Oklahoma State University Stillwater, OK 74078 KEY PERSONNEL. See Name Chang-An Yu Linda Yu Li Zhang Lian-Quan Gu Sudha Shenoy Johann Deisenhofer Francis Millett Mario Rivera PHS 398 (REV. 4/98) Number pages consecutively instructions on Page 11. Use continuation pages as needed to provide the required information inthe format shown below. Organization Role on Project Oklahoma State University P.I Oklahoma State University Co-Pi Oklahoma State University Senior Res. Associate Oklahoma State University Visiting Professor Oklahoma State University Res. Associate University of Texas, Southwestern Medical Center Collaborator University of Arkansas Collaborator Oklahoma State University Collaborator Page 2 BB~ at the bottom throughout the application. Do notuse suffixes such as 3a, 3b. cc pstigator/Program Director (Last, first, middle): Yu, Chang-An Type the name of the principal investigator/prog fain director at the top of each printed page and each co: uation page. (For type specifications, see instructions on page 6.) RESEARCH GRANT TABLE OF CONTENTS Page Numbers Face Page 1 Description,